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Rare earth-based Parallume optical encoding technology can be used to multiplex arbitrarily large numbers of samples. The Parallume materials are non-toxic and chemically inert, cannot be photobleached and require only a single LED excitation source.

The rare earth-based Parallume optical encoding materials enable for the first time the multiplexing of arbitrarily large numbers of samples including assays based on nucleic acids or protein-antibody pairs or the multiplexed immunohistochemical staining of cells and tissues.


A multiplexed assay refers to the simultaneous screening within a single container of a multicomponent Target mixture against a large number of probes each of which gives information about a particular constituent or component of the target mixture. The hybridization of a DNA microarray with 1000 probes against a single target mixture is a multiplexed assay whereas an assay performed separately in 1000 microtiter plate wells is not. The multiplexing of N sample assays has many advantages over serial high throughput protocols including:

Parallume Encoded Agarose Beads
Fig. 1 A photomicrograph of Parallume-encoded beads under 320 nm excitation. In optical multiplexing, a given probe is attached to a bead with a unique optical signature allowing reactions of that probe to be optically distinguished from other beads possessing other probes when the beads are pooled.
  • Benefits such as reduced costs, fewer containers, less sample handling and shorter time to answer - these savings increase as the multiplexing depth increases
  • An N-fold more concentrated target sample and therefore N-fold better detection limits as compared to diluting the target sample for N individual assays
  • All assays are performed under exactly the same conditions
  • Ability to include a large number of different controls
  • Lower reagent usage and less waste generated

Optical Multiplexing

The identity of each probe used when multiplexing with microarrays is known from its geometric location within the array but this requires loading the probe onto a precisely known location on the substrate. In optical multiplexing, the probes are attached to encoded beads or nanoparticles, which are loaded randomly into the assay container without regard to the order, sequence or timing of addition, and the identity of each probe determined from a unique optical signature emitted from each bead or nanoparticle (Fig. 1). The optical signature may be based on either measurement of (a) the absolute intensities of the various emitter colors or (b) ratios among emitter colors.

The most desirable and useful optical multiplexing platform technology would include:

  • Thousands or millions of resolvable optical codes to allow arbitrarily large numbers of samples to be assayed
  • Substrates, reagents and protocols that are compatible with any protein or nucleic acid assay
  • Codes that cannot be changed by chemicals or photobleaching when measured
  • Optical codes that are invariant with respect to bead size, brightness of excitation source, angle of illumination
  • Encoded beads or nanoparticles sets which may be handled in conventional laboratory apparatus
  • The optical code is generated from a single excitation unlike organic dyes which require a separate source for each dye
  • No optical crosstalk or interference between emission from the optical encoding material and reporter emission
  • Rapid, accurate and high throughput protocols and instruments for collecting and analyzing the multiplex data

The fact that current multiplexing technology, which is based on mixtures of organic dyes, is limited to a few dozen optical codes is primarily a result of the broad emission peak widths. This spectral overlap, rapid photobleaching and the requirement for a separate laser excitation source for each dye color prevents accurate determination of the relative brightness of two emitters. Recent work on optical encoding with quantum dots has shown that, in addition to the toxicity and difficulty in conjugating biocontent, it not possible to create more than a few optical codes with quantum dots because of large changes in emission intensity with pH, buffer, unstable emission, etc. which are different for each quantum dot color. The Parallume optical encoding technology presented here directly addresses these issues.

Optical Multiplexing with Parallume Materials

The Parallume encoding materials, which are based on exceedingly stable, solid state inorganic oxides containing multicolor rare earth emitters, are essentially immutable and cannot be photobleached [n2], oxidized or dissolved and emission is independent of any environmental factors. Very importantly, the Parallume materials are excited and emit all colors using a single excitation source. The emission spectrum of a typical Parallume optical encoding material is shown in Fig. 2 and illustrates that emission from the multiple Parallume rare earth emitters (peak width = 2-10 nm FWHM) is far narrower than that from typical organic dyes or quantum dots (e.g., 28nm for QD625, 33nm for Cy5, 46nm for Oregon Green and 65nm for QD705FWHM).

Peak width of fluorescent emission, organic dyes, quantum dots, and a single 5-emitter Parallume composition
Fig. 2 A comparison of the peak width of the fluorescent emission from organic dyes, quantum dots and a single 5-emitter Parallume composition. The emission peaks of the rare earth Parallume encoding materials display much less spectral overlap than organic dyes. This allows orders of magnitude more optical codes to be resolved with Parallume encoding than is possible when encoding with dyes or quantum dots.

The near baseline resolution of the different Parallume rare earth emitters allows very accurate determination of the relative fluorescent intensity of the emitters resulting in a far larger number of available optical codes than any other current optical multiplexing technology.

Table 1 summarizes the features, advantages and benefits when using Parallume optical encoding materials compared to dyes or quantum dots.

Table 1: Advantages of Parallume Encoding Materials

Property or Feature
Use of narrow band rare earth fluorophores Millions of resolvable optical codes
Cannot be photobleached Codes stable indefinitely with no change
Cannot be oxidized or dissolved Stable to > 1500°C
All colors emitted from single excitation source Single LED excitation source required for Parallume
Ratiometric optical codes Optical code independent of particle or bead size and brightness of illumination source
Chemically inert Similar toxicity to aluminum oxide or silica
Beads or nanoparticles easily dervitized Any nucleic acid, protein or small molecules may be covalently attached to encoded particle
Facile bulk commercial synthesis Inexpensive, scalable and reliable supply

Ratiometric Optical Codes

In some optical encoding technologies, every bead must be exactly the same diameter (volume) because absolute intensity measurements are needed. For example, some optically encoded beads for flow cytometry must be exactly the same size which results in a very high cost. Because the intensity of the multicolor, narrow band Parallume rare earth emission peaks may be measured so accurately, Parallume encoding can create optical codes based onthe intensity ratios of colors instead of their relative absolute intensity as illustrated schematically in Fig. 3. The optical code is now independent of bead or particle size and brightness of the excitation source.

Parallume encoding uses optical bins on ratiometric instead of absolute intensity measurements
Fig. 3 Parallume encoding uses optical codes based on ratiometric instead of absolute intensity measurements to eliminate difficulties in code rendering from changes in sample size, excitation source brightness or angle of illumination. The hexanary oxide (1) emits six different colors (left) whose relative intensities are schematically illustrated (center). Since we can resolve >100 binary ratios between two colors (see below) the 6-color Parallume system shown can yield ten billion resolvable optical codes.

Ten Billion Parallume Optical Codes

Using a ternary Parallume system as an example, Fig. 4 shows there are at least 100 resolvable ratios for binary color pairs. As shown in Fig. 3, there are around 10 billion resolvable Parallume optical codes for six colors and a 1% compositional resolution limit.

Fig. 4 The ratio of emitted light (inset) to the molar ratio of two emitters is linear and is shown for selected compositions from the ternary Dy-Er-Sm phase diagram. The Dy, Er and Sm emitter amounts are given as a percentage of their total a, b and c in the Parallume material of formula Y1-x[DyaErbSmc]xVO4 where a = c = 0.001 and b = 0.002. The data indicates that there are at least 100 resolvable ratiometric optical codes for each emitter pair or MN-1 optical codes for M resolvable binary ratios and n different emitter colors. Inset: Tm, Sm and Er Parallume nanoparticles suspensions (302nm excitation).

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